Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
  • C12N9/82—Asparaginase (3.5.1.1)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/88—Serratia
  • Y10S435/881—Serratia marcescens

Definitions

• the invention relates to a process for producing L-asparaginase by cultivating L- asparaginase-producing micro organisms belonging to the genus Serralla in a suitable nutrient medium while maintaining a control on the amount of ammonia nitrogen in the medium.
• one of the objects of the present invention is to provide an improved process for the production of L- asparaginase which overcomes the disadvantages and deficiencies of the prior art methods.
• Another object of the present invention is to provide a process for producing L-asparaginase by fennentation which may be carried out in an efficacious and relatively simple manner.
• a further object of the invention is to provide a process for producing L-asparaginase by fermentation which may be carried out advantageously on a large scale to give a good yield of product.
• a still further object of the invention is to provide 1..- asparaginase.
• the present invention is also concerned with the production of L-asparaginase having an antitumor activity by cultivating a micro-organism belonging to the genus Serratia in a liquid culture medium instead of the conventional solid culture medium, as in the aforementioned patent application.
• the antitumor L- asparaginase cells can be produced in greater yields by using a relatively high concentration of carbon source in the culture medium while properly checking the amount of nitrogen present in the form of ammonia (NH,,N).
• the other nutrients such as a carbon source, inorganic salts and the like
• the other nutrients such as a carbon source, inorganic salts and the like
• the amount of nitrogen in the form of ammonia in the culture medium varies in dependence upon the nitrogen source (organic and inorganic materials) used together with the carbon source and the like.
• the amount of nitrogen source present in the medium is large, the growth of the micro-organisms is good.
• the cultivation is carried out by keeping the amount of ammonia nitrogen at a fixed level mg./ml. or less), the formation of the desired cells is favorable.
• micro-organisms belonging to the genus Serratia which are capable of producing L-asparaginase can be used in the present invention.
• micro-organisms belonging to Serrazia marcescens are particularly preferred.
• the micro-organism is inoculated into a culture medium containing a 1-15 percent concentration of carbon source (carbohydrates such as glucose, sucrose, fructose, glycerol and the like) and further containing an appropriate amount of the nitrogen source (including organic and inorganic materials), inorganic salts and other nutrients. Thereafter, the cultivation is conducted by maintaining the amount of nitrogen in the form of ammonia in the culture medium at a concentration of 10 mg./ml. or less and preferably at 5 mg./m
• Process A Cultivation under aeration and with agitation for 16 hours at 30 C. in a culture medium of pH 7.0 containing 0.3% glucose, 1.0% meat extract, 1.0% peptone, 0.5% sodium chloride and 0.5% yeast extract [from Japanese Application No. 9116/1958).
• Process B Same as Process A except that the amount of glucose is 4.0% and the pH is adjusted to 7 .08.5 with ammonia water [from Japanese Application N 0. 9116/1968].
• Process 0 Cultivation under aeration and with agitation for 36 hours at 30 C. in a culture medium containing 4.0% glucose, 2.0% meat extract, 2.0% peptone, 0.5% yeast extract and 0.5% sodium chloride. The pH of the medium is adjusted to 7.0-8.5 with sodium hydroxide ⁇ from the present invention].
• the obtained L-asparaginase preparations show an antitumor activity which is capable of completely curing an experimental leukemia of a mouse similarly as in the case of the aforementioned patent application.
• Either a synthetic culture medium or a natural nutrient medium is suitable for cultivation of the strains employed in the present invention as long as it contains the essential nutrients for the growth of the strain employed.
• Such nutrients are well known in the art and include substances such as a carbon source, a nitrogen source, inorganic compounds and the like which are utilized by the micro-organism employed in appropriate amounts.
• a carbon source there may be mentioned, by way of example, carbohydrates such as glucose, fructose, maltose, sucrose, starch, starch hydrolysate, molasses, etc., or any other suitable carbon source such as organic acids, for example, acetic acid, lactic acid, etc. These substances may be used either singly or in mixtures of two or more.
• various kinds of inorganic or organic salts or compounds such as urea, liquid ammonia or ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, ammonium phosphate, etc, or natural substances containing nitrogen, such as comsteep liquor, yeast extract, meat extract, peptone, fish meal, bouillon, casein hydrolysates, casamino acid, fish solubles, rice bran extract, etc. may be employed. Again, these substances may be used either singly or in combinations of two or more.
• Inorganic compounds which may be added to the culture medium include magnesium sulfate, sodium phosphate, potassium dihydrogen phosphate, potassium monohydrogen phosphate, iron sulfate, manganese chloride, calcium chloride, sodium chloride, zinc sulfate, etc.
• Growthpromoting agents such as biotin, or vitamins, such as thiamine or cobalamin, may also be added to the medium if desired or required.
• the fermentation or culturing of the microorganisms is conducted under aerobic conditions, such as aerobic shaking of the culture or with stirring and aeration of a submerged culture, at a temperature of, for example, about 20 to 40 C. and at a pH of, for example, about 6.0 to 10.0. After about I to 3 days of culturing under these conditions, large amounts of L- asparaginase are found to be accumulated in the resultant culture liquor.
• the L-asparaginase can be removed by conventional means, such as ion exchange resin treatment, extraction with solvents, precipitation, adsorption, chromatography or the like.
• Serratia marcescens ATCC-60 is cultivated in a liquid nutrient medium containing 2 percent of dry bouillon with aeration and agitation for 7 hours at 30 C.
• the resultant culture liquor is used as the seed culture.
• This seed culture is inoculated in a jar fermentor having a capacity of liters in the proportion of 10 percent by volume into 3 l. of a fermentation medium having the following composition:
• the cells are suspended in a 0.01 M tris-buffer solution (pH 8.5) and treated with a Sonic Oscilator 10 Kc) for 10 minutes in order to destroy the cells.
• a crude extract liquid having an L-asparaginase activity of 150 units/ml. and a specific activity of 25.0 units per 1 mg. of protein is obtained.
• This extract liquid is subjected to a nucleic acid-removing treatment with Mn, removal of impure proteins by heating, fractional precipitation with ammonium sulfate, chromatography with diethylaminoethyl cellulose and biogels and the like, whereby the enzymatic protein is purified to give an L-asparaginase preparation having a specific activity of 1500 units per 1 mg. of protein in a yield of about 20 percent.
• This preparation shows an antitumor activity which is capable of completely curing an experimental leukemia with a dosage of several 10's of pg.
• EXAMPLE 2 Serralia marcescens ATCC l9l80 is cultivated in the same manner as described in example 1 in order to prepare a seed culture.
• This seed culture is inoculated in a jar fermentor having a capacity of 5 liters in the proportion of 10 percent by volume into 3 liters of a fermentation medium having the following composition:
• the resultant cells are separated by a centrifugal separator to obtain 360 grams of wet cells. These cells are subjected to the same treatments as described in example 1 for purifying the enzymes, whereby a preparation having a specific activity of 2000 units per 1 mg. of protein is obtained in a yield of about 20 percent. This enzymatic preparation is effective against leukemia in a mouse similarly as in example 1.
• EXAMPLE 3 Serratia marcescens KY 4l04 ATCC 13880 is cultivated in the same manner as described in example 1 in order to prepare a seed culture.
• This seed culture is inoculated in a fermentation tank having a capacity of 2 KL in the proportion of i0 percent by volume into i KL of a fermentation medium having the same composition as described in example 2.
• Cuituring is then carried out at 30 C. for 60 hours with aerobic shaking of the culture at r.p.m. and with aeration at the rate of 400 liters per minute.
• the pH value of the medium is adjusted to 7.0-8.5 with a 40 percent solution of potassium hydroxide during the cultivation.
• the resultant cells are separated by a centrifugal separator, thereby obtaining I30 kg. of wet cells.
• the obtained cells are treated in the same manner as described in example 1, whereby a crude enzymatic liquid having an L-asparaginase activity of 198 units/ml. and a specific activity of 27.6 units per i mg. of protein is obtained.
• the crude liquid is further purified in the same manner as described in example 1, resulting in an L-asparaginase preparation having a specific activity of 2000 units per 1 mg. of protein in a yield of about 25 percent.
• This enzymatic preparation is effective against leukemia in a mouse in the same way as discussed in example I.
• L-asparaginase which comprises culturing an L-asparaginase-producing micro-organism belonging to the genus Serratia under aerobic conditions in an aqueous nutrient medium containing about i to 15 percent by weight of carbon source and recovering said L-asparaginase from the resultant culture liquor, the improvement which comprises maintaining the amount of nitrogen present in the medium in the form of ammonia at 10 mg./ml. or less.
• micro-organism is Serratia marcescens.
• a process for producing L-asparaginase which comprises culturing an L-asparaginase-producing micro-organism belonging to Serratia marcescens under aerobic conditions in an aqueous nutrient medium containing at least a carbon source in the amount of about 1 to percent by weight and a nitrogen source, the amount of ammonia nitrogen in the medium being maintained at 10 mg./ml. or less, and recovering an L-asparaginase preparation from the resultant culture liquid.
• micro-organism is Serratia marcescens ATCC 60.
• micro-organism is Serratia marcescens ATCC 19180.
• micro-organism is Serran'a marcescens KY 4104 ATCC i388.
• a process for producing L-asparaginase which comprises culturing a micro-organism selected from the group consisting of Serratia marcescens ATCC 60, Serrau'a marcescens ATCC 19180 and Serratia marcescens KY 4104 ATCC 13880 under aerobic conditions in an aqueous nutrient medium containing at least from 1 to l5 percent by weight of a carbon source and a nitrogen source, the amount of ammonia nitrogen in the medium being maintained at 5 mg./ml. or less, and recovering an L-asparaginase preparation from the resultant culture liquid.

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• Enzymes And Modification Thereof (AREA)
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• Preparation Of Compounds By Using Micro-Organisms (AREA)

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• 1969
  • 1969-03-13 CA CA051,374A patent/CA940852A/en not_active Expired
  • 1969-05-21 DE DE1925952A patent/DE1925952C3/de not_active Expired
  • 1969-05-27 US US828332A patent/US3627639A/en not_active Expired - Lifetime
  • 1969-05-29 GB GB27343/69A patent/GB1215786A/en not_active Expired
  • 1969-05-30 FR FR6917688A patent/FR2010156A1/fr not_active Withdrawn

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